Because of their selectivity and high level of catalytic activity, enzymes are increasingly being used in many sectors of the food, pharmaceutical and chemical industries, for the production and analysis of products, as well as in medicine for diagnosis and therapy. Although enzymes are not used up in the conversion which they catalyze, they cannot be reused, because of their substrate solubility. This brings a number of disadvantages with it, and attempts have been made for quite some time to overcome these by using immobilized enzymes.
Immobilization is understood to be the transformation of water-soluble enzymes into a form insoluble in water, while maintaining their catalytic effectiveness. This is possible by chemical and/or physical binding of the water-soluble enzymes to a carrier insoluble in water, as well as by inclusion in gel matrices or microcapsules which are insoluble in water. The use of immobilized enzymes is limited, as a matter of principle, to processes with aqueous substrates or liquid substrates that contain water. The significant advantages of immobilized enzymes consist of the ease in separating them from the reaction solution and in the fact that they can be reused. These advantages result in significant cost savings, particularly in the case of enzymes which are not easily accessible and can be produced only in small yield. Since the end products remain free of enzymes, the heat treatment for inactivation of dissolved enzymes, which is necessary otherwise, is also eliminated, which is particularly advantageous in the case of heat-sensitive products. In addition, it is possible to use a continuous process with precise process control when using immobilized enzymes.
Every method with immobilized enzymes is in competition with the same method with dissolved enzymes. Immobilized enzymes are only competitive if clear economic advantages can be achieved with their use, for example in that improved and purer products are obtained, which can be processed more easily, faster and at a lower cost.
For the immobilization of enzymes, the following methods have been known:
adsorption PA1 ionic binding PA1 absorption PA1 covalent binding to a carrier surface PA1 inclusion in a matrix or in microcapsules PA1 inclusion by sheathing with a membrane (macroencapsulation) PA1 cross-linking or copolymerization with difunctional or polyfunctional monomers.
However, all of these methods are not universally applicable. Only when the application of an enzyme has been precisely defined can a suitable carrier, the immobilization method and the reactor form be selected and coordinated with one another (see, for example: W. Hartmeier, "Immobilisierte Biokatalysatoren" ["Immobilized Biocatalysts"], Springer-Verlag Berlin, Heidelberg 1986, pages 23 to 51, and J. Woodward, "Immobilised cells and enzymes", IRL Press, Oxford, Washington D.C., 1985, pages 3 to 54, as well as W. Crueger and A. Crueger, "Biotechnologie--Lehrbuch der angewandten Mikrobiologie ["Biotechnology--Handbook of Applied Microbiology"], R. Oldenbourg Verlag Munich, Vienna 1989, pages 201 to 203).
Physical adsorption of an enzyme on a carrier insoluble in water is the simplest and oldest method for immobilization of enzymes. It is based on non-specific physical interactions between the enzyme protein and the surface of the carrier material. The binding forces are mainly hydrogen bridges and van der Waals forces (see in this regard: S. A. Barker and I. Kay in "Handbook of Enzyme Biotechnology" (Editor: A. Wiseman), Ellis Horwood, Chichester 1975, Chapter 5, page 89). For immobilization, a concentrated enzyme solution is mixed with the carrier material carrier materials often used are activated charcoal, aluminum oxide, silicon dioxide, porous glass, cellulose and phenolic synthetic resins.
Adsorption has the disadvantage that because of the weak binding forces, desorption of the enzyme occurs over the period of use, by changes in temperature, pH or ionic strength, or due to the presence of other substances in the reaction solution. Another disadvantage is that adsorption is not specific, and thus further proteins or other substances can be adsorbed from the reaction solution. This can cause changes in the properties of the immobilized enzyme, and activity losses can occur.
In the case of ionic binding, the electrostatically charged enzyme molecule is attracted and fixed in place by a polyanionic or polycationic carrier with the opposite charge. As in the case of adsorption, again only a relatively weak bond occurs, since the charge of the enzyme protein is very small relative to its mass. The use of this method is also only possible for very low salt contents of the substrate solution, since other stronger ions can easily displace the enzyme molecules if they are present in the substrate. The ion exchanger resins which are most frequently used are DEAE cellulose (DEAE=diethylaminoethyl), DEAE Sephadex (an agarose preparation), and CM cellulose (CM=carboxymethyl). Also in the case of absorption in polymer layers, relatively unstable systems are obtained. Migration and extraction of the enzymes result in a constant decrease in activity and limit the lifetime of the enzyme layer.
Significantly more stable systems are achieved if the enzymes are covalently bound to a carrier surface, made insoluble via cross-linking or copolymerization, or are immobilized by microencapsulation or macroencapsulation. For the formation of covalent bonds and for cross-linking, free amino, carboxyl, hydroxyl and mercapto groups are available on the part of the enzymes. Both inorganic materials, such as glass, and natural and synthetic organic polymers can be used as the carrier material. A prerequisite in this connection is that the carrier materials contain reactive groups, such as isocyanate, isothiocyanate, acid chloride and epoxy groups. Less reactive groups can be activated, for example carboxyl groups can be activated using the carbodiimide or azide method, hydroxyl groups can be activated using the bromine cyan method, and amino groups can be activated using the isothiocyanate or azo method. It was possible, particularly on the basis of acrylic acid and methacrylic acid derivatives, to produce numerous reactive copolymers with dinitrofluorophenyl, isothiocyanate, oxirane or acid anhydride groups. Polyacrylamides with oxirane groups as well as modified copolymers on the basis of vinyl acetate and divinyl ethylene urea with oxirane groups are commercially available, for example.
Immobilization by cross-linking or by copolymerization represent special forms of covalent binding. In these methods, the formation of covalent bonds takes place between the enzyme molecules and difunctional or polyfunctional monomers, such as glutardialdehyde, or, in the case of copolymerization, additionally between the enzyme molecules and a polymerizing substance. In this manner, insoluble aggregates with a high molecular weight are formed. Cross-linking is generally used as an immobilization method in combination with one of the other methods, for example in combination with adsorption or absorption. Here, the enzyme molecules are first adsorbed on the surface of the carrier, or are absorbed in a layer located on it, and subsequently cross-linked.
A significant disadvantage of immobilization by covalent binding is the great stress on the biocatalysts connected with it. The immobilization procedures that are necessary, some of which are rough, in which a strong change in the pH occurs, organic solvents have to be used or reaction with reactive substances with a low molecular weight takes place, almost always lead to strong conformation changes and thus to activity losses of enzymes bound in such manner.
In immobilization by inclusion, i.e., microencapsulation or macroencapsulation, the enzymes themselves are not made insoluble, rather their reaction range is limited by semipermeable polymers or polymer layers. A prerequisite for the ability of enzymes sheathed in this manner to function is that substrates and products can pass through the sheathing substance, while the enzymes themselves have to be held back. In addition to natural polymers, such as alginate, carrageenan, pectin, agar and gelatin, which are, however, too large-meshed for permanent immobilization of enzymes, synthetic polymers, such as polyacrylamide, are particularly used for matrix sheathing. Polyamides, polyurethanes, polyesters and polyureas, for example, are used for encapsulation. The inclusion method has the disadvantage that relatively thick layers with long diffusion paths are formed.